We propose to utilize our recently developed microfiltration technique with an enzyme labeled staphylococcus Protein A (SPA) assay to test for ocular herpes simplex virus (HSV) and varicella zoster virus (VZV) antigens on cells taken directly from clinical lesions. Preliminary data using 125I-SPA and microfiltration techniques indicate that this assay is sensitive, rapid and uniquely specific. We will attempt to substitute enzyme labeled SPA for 125I-SPA in order to make the assays optically readable. We intend to make this assay simple enough to be used by all clinical laboratories. We will establish, standardize and compare these SPA assays to ELISA assays using both tissue culture and rabbit model systems. The best of these methods will then be evaluated on human clinical material in comparison to direct immunofluorescences and viral isolation techniques. We will also use this microfiltration methodology with SPA assays to detect and quantitate class specific (IgA, IgG and IgM) anti-herpes virus antibodies in serial samplings of patient lacrimal fluid and serum. The results of these studies will be correlated with viral isolation and clinical manifestations to determine if they are of diagnostic or prognostic significance. In addition, we will investigate the pathogenesis of recurrent HSV stromal keratitis in human corneas scarred by recurrent disease. We will perform correlative studies on corneas from patients with a history of recurrent disease or in cultured cells derAved from these corneas. These studies will determine the presence or absence of (1) infectious HSV (by direct virus isolation), (2) latent HSV (by co-cultivaton), (3) HSV particles (by electron microscopy), (4) HSV genetic information (by conventional DNA and mRNA hybridization and in situ DNA hybridization), (5) HSV antigens (by SPA microfiltration assays and by immunoperoxidase staining techniques), (6) an inflammatory response (by histological examination). The objective of these studies is to understand the pathogenesis mechanism(s) of the disease so that we can more effectively manage or eliminate the morbidity of recurrent HSV keratitis.